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We are purifying a bacterial dehydrogenase from E. coli cells using a histidine tag. We purified the same dehydrogenase from different bacteria previously, and we have a few examples from the literature. The dehydrogenase from this organism is at least 100-fold lower in specific activity, perhaps more than that. We have a little experience with nickel columns, but not a great deal. Could a small amount of nickel ion be inhibiting our protein? We will try an experiment in which we incubate the enzyme with DTT shortly (there is an essential cysteine). What are other possible explanations?

We just tried the "Enzyme Kinetics" software module for the first time on a Cary UV/VIS spectrophotometer, which is separate from the Kinetics module. We are plotting absorbance of NADPH versus time. One of its features is that it can calculate the slope of the trace just after one adds the final component to the cuvette holding buffer and all other components. The user defines the time period, which is the linear portion, and the software provides the slope (the units of the slope are a little bit mysterious). This feature could save a good deal of time when one has multiple runs, but there are a couple of software...curiosities. The first is that the software asks for the micromolar (not the molar) absorptivity. IIUC this is 0.00622 per µM per cm. The second is that when we try to input this number, the software increases the value, to 0.01 if I am not mistaken. We tried writing the number in a type of scientific notation, but to no avail. Has anyone used this software module? If so, how does one put in the correct value of the absorptivity? If worse comes to worse, we will just do the slope calculation ourselves.

In the past I followed a protocol based on Methods in Enzymology Volume 174. We warmed the tubing a bicarbonate/EDTA buffer to about 80 °C. We rinsed several times with warm DI water. We stored in the presence of a preservative, often sodium azide. Is this similar to what other labs do? EDT Does anyone have experience with SnakeSkin dialysis tubing? It seems to be much less expensive than the alternative, although that may be because of a promotion.

I probably do not understand your question. We were planning to use a classical gradient maker and gravity to run this column, as opposed to FPLC. However, we have access to an FPLC, and we have a few anion-exchange columns. I was thinking that this might be an appropriate second purification step, if one were needed. Cibacron Blue chromatography might also be a good second step.

We would like to purify this protein away from others for kinetic and structural studies. We anticipate that there will be a buffer exchange step afterwards to remove the high concentration of imidazole.

I saw a paper one time that indicated a much better expression in one cell line versus another. It might have been BL21 versus B834. If you are still having problems, I will try to turn up the reference.

We are following a paper in which they purified their histidine-tagged protein (same protein as ours) using a gradient of imidazole. However, they did not specify the volume of buffers. This affects the steepness of the gradient. What would be a logical choice? A Methods in Enzymology article from around 2000 by Barnhorst and Falke Reference suggested a 20-column volume gradient.

I found a paper which expressed the same protein from the same organism as we are trying to do. They uses BL21(DE3) star, and a pET-based plasmid. What struck me is that their conditions for expression (10 µM IPTG; 23 °C for twelve hours) are quite a bit different from any that I have previously used. In their paper they discuss two proteins, and they indicate that they chose conditions to minimize inclusion body formation and to increase yield of protein. I can see the logic in not making inclusion bodies; this protein exists as a dimer, and the monomers are around 35-40 kilodaltons (I have refolded small, monomeric proteins from inclusion bodies, but I don't think that this would be my first choice here). Should we go with their conditions, or should we try 30° and 0.5 mM IPTG, which worked for their other protein and which are closer to other conditions that I have seen?

Does anyone sell electrocompetent cells for transformation by electroporation? Or is it just better to make one's own?

When you use a metal loop, do you put a curve or bend into it. I found myself digging a little bit into the agar as I was streaking.

CharonY, Thank you so much for a thoughtful reply. For better or worse, I have to be the teacher in this project. We poured LB-kanamycin plates today, and it seemed to go reasonably well. We will try streaking plates tomorrow. BTW I found this video on pouring plates to be a helpful reminder to me, and I plan to look for more such videos.

I left the field of protein biochemistry to pursue a project in synthetic chemistry some years ago, and now this project is veering into protein expression and purification. We ordered two plasmids from Genscript, and they arrived in the form of what appear to be stab cultures. Therefore, we need to move forward with deliberate speed. I have a basic understanding of sterile technique, but my skills are rusty. What are reliable sources of information regarding handling of E. coli? Now, we need to store our strains, which arrived as stab cultures. In the future, we may need to work out details of the purification of our enzyme, because it is coming from different organisms versus previously reported one. I have a good knowledge of classical protein chromatography but not medium-pressure chromatography. How should I get up to speed in this area? I realize that my questions are broad.

I decided to try a test run today. The stacking gel is only a few millimeters in length. I started at 150 volts and about 60 milliamps, and it seemed as if the current decreased over time. The gel ran more quickly than what I was used to. I am destaining the gel now, and the bands look good.

I am trying precast gels for the first time, and it is for a laboratory class. I talked to someone in technical services, and he sent a pdf with some instructions. As I read them, I did not see anything about changing the voltage between the stacking gel and the resolving gel. I will try something similar to what you suggest.

I have cast and used SDS PAGE with Tris/glycine/SDS electrode buffer. However, I was planning to use some precast, gradient gels for the first time. I am trying to contact the manufacturer, but I have not yet heard back. "BisTris" is in the product description, and the catalog page mentions the possibility of ordering MES or MOPS buffer. My first question is whether I should change my recipe for the sample loading buffer. My old recipe includes Tris (pH 6.8, same as stacking gel), SDS, 2-mercaptoethanol, glycerol, and bromphenol blue. My second question is what sort of power settings should I use? In the past I have typically used about 100 volts during the stacking portion of the run, which changes to 150 volts or more volts once the samples enter the resolving gel.

I tried the option of print to pdf today. I chose the landscape option. I still cannot fill more than about half the page with the spectrum. On the other hand, I was able to get better results with Microsoft's Snip tool than I had previously: I resized the scan within the Cary software. I will keep working on this.Fraction_17_gelfiltration_landscape.pdfFraction_17_gelfiltration_landscape.pdf

Thank you; this is helpful in a couple of ways. One, it notes that one cannot directly export the spectrum as a jpg or png, which is what I thought. Two, it confirms that the spectrum can be exported as a csv file. The problem with csv files is that they take a bit of editing before one gets a usable spectrum (this is not ideal in a classroom application).

After a long hiatus, I am using Cary spectrophotometers again. I am struggling to find the best way to export just a spectrum electronically, especially an electronic format that could be imported into a Word document. As a work-around, I used Microsoft's Snip and Sketch tool (not sure that this is the correct name) to make .jpg or .png files, but this is an imperfect solution. When I tried to export the Report as a .pdf file, I obtained the spectrum plus parameters, and for right now, it is just the spectrum that I want. I tried two other formats, .DSW and .RTF, but I was not successful. The details escape me at the moment, but I will try to return to this question later. For a very different program I used .wmf format (Windows Meta File), but that is not available in this instance. Any thoughts? Thank you in advance.

The assays that I have in mind are disk diffusion assays and assays to determine minimum inhibitory concentration or related. Sometimes these assays are conducted in rich medium (Luria broth, Terrific Broth, or similar). It seems to me that this choice biases against finding inhibitors that target enzymes whose absence is conditionally lethal. If one inhibits an enzyme that produces a metabolite that can be obtained from the medium, then growth of the cells might be impaired little or not at all. If one is interested in developing an antimicrobial compound, perhaps using a medium that mimics human serum is a better choice, but I imagine that there are complications I doubt very much that I am the only person who has wondered about this possible problem. Are there any papers that touch upon this subject or address it in a systematic manner.

Faucher and Maitre Synthetic Communications 2003 33(20):3503-3511. DOI:10.1081/SCC-120024730 I would consider the possibility that TCEP is reducing DMSO, although the presence of iodine may be necessary for a rapid reaction (see Faucher and Maitre). If I could find no more information on this topic, I would consider switching my solvent to DMF. Is there any reason that you are using TCEP and not 2-ME or DTT as your reducing agent?

Tutoring is not a replacement for studying on your own, IMO. It can fill in the gaps when you get stuck. My advice is to turn off all electronic distractions; short, intense studying is more effective than long periods of diffuse studying.

If I recall correctly I once used bromocresol green to detect compounds containing a carboxylic acid group on a TLC plate. That is probably different from the sort of reaction that you were thinking about.

A catalyst such as thiamine pyrophosphate can decarboxylate alpha-keto acids.

Hi CharonY, Thank you for a very helpful answer. We used established antimicrobial substances as positive controls; we were not comparing our compounds to them in any other sense.

Many covalent inhibitors are irreversible, but not all of them are. If each step to form a covalent bond is reversible, then the whole process is reversible. A good example is peptide aldehyde or trifluromethylketone binding to the nucleophile in a cysteine or serine proteases. Reversible, covalent inhibitors seem to be catching on, so it's a good topic.